The growth of long bones and the endochondral cranial base is generated by spatial and temporal control of chondrocyte maturation in the growth plate cartilage. This maturation process gives rise to domains with distinct cell morphologies and gene expression profiles. Genetic studies provide evidence that proper domain architecture is crucial to growth plate function, and this architecture is susceptible to damage via injury or disease. One important event in chondrocyte maturation is the reorganization of round precursor cells into discoid proliferative chondrocytes that form columns via clonal expansion. Previous studies support a model in which column formation by proliferative chondrocytes occurs through sequential planar division and cell intercalation in a manner similar to convergent extension. One important caveat is that these studies, and the proposed models of cell behavior in the growth plate, are based on the analysis of static images from sections of fixed tissue. The absence of dynamic information reduces confidence in the models and places limits on the mechanistic interpretation of genetic data. This proposal utilizes an in vitro live imaging system recently developed in the laboratory to elucidate the mechanisms that regulate column formation in proliferative chondrocytes. Specifically, the aims are designed to test the hypothesis that column formation occurs by cell intercalation in a convergent extension process that is driven by interactions at the interface of daughter cells. This hypothesis will be tested using live imaging, together with genetic and chemical-genetic methods, to detect the presence of lamellopodia in daughter cells, to determine if actin dynamics and integrin function are required for column formation, and to determine if premature separation of daughter cells prevents cell rearrangement in Piga mutant proliferative chondrocytes. Results of these studies will provide important new information for the generation of cartilage architecture by tissue engineering and will establish this innovative method as a crucial new tool for study of the mechanisms of growth control.